qPCR Sets New Standards for Gene-Expression Analysis

5’ FAM probes with five different quenchers: Each probe was synthesized to target the ACTB locus and all were run in triplicate with the same primer products and 0.5 ng of cDNA. All reactions were run under standard cycling conditions. [Integrated DNA Technologies]

Quantitative real-time polymerase chain reaction (qPCR) continues to gain popularity as the premier technology for gene-expression analysis. While new strides are being made to refine and improve qPCR, problems remain—especially the need to improve consistency and build confidence in data analysis. Select Biosciences’ “qPCR Europe” conference held last month in Dublin addressed current issues and highlighted emerging tools and strategies for this continuously evolving field.

Mark Behlke, M.D., Ph.D., CSO of Integrated DNA Technologies reported on the company’s new technology to enhance the performance of qPCR. “A central strategy common to many qPCR methods is the use of complementary hybridization probes bearing a 5´-dye and a 3´-quencher.

“Following hybridization with the target, the probe becomes degraded and the amount of fluorescence generated is proportional to how much target is present. Sensitivity is improved by having a high signal-to-noise ratio. Good quencher performance will help lower background, improving sensitivity and assay performance. But often, this can be a challenge.”

According to Dr. Behlke, many strategies have emerged over the years to enhance qPCR sensitivity. “Quenchers are normally placed at the 3´ end of the probe, which is usually 25–30 bases away from the fluorescent dye. One strategy to improve the quality of quenching is the use of molecular beacons that have hairpin handles that fold the probe, forcing the dye and quencher to be closer.

“Another strategy is to place the quencher internally within the probe; however, this approach usually destabilizes hybridization and can hurt probe performance instead of improving it. The use of Tm-enhancing chemical modifications allows probes to be shorter, thereby improving quenching, but these methods always increase cost.”

IDT recently developed a new double-quenched probe design that employs a dark quencher (ZEN™) that is inserted internally within the probe, 9–10 bases from the 5´ fluorophore. The design also keeps the standard 3´ quencher, which, in addition to quenching, also blocks the 3´ end and prevents the probe from acting like a primer.

“The new probe design is particularly effective in enhancing sensitivity and reducing background,” Dr. Behlke explained. “Importantly, the ZEN quencher actually stabilizes probe hybridization to target, increasing Tm. This is quite novel, as all other quenchers we tested were destabilizing when inserted in the middle of a probe. The other piece of good news is that the ZEN modification is a robust chemistry and is surprisingly straightforward to manufacture, allowing us to make our dual-quenched ZEN probes with no significant cost increase.

The company is utilizing the new technology in its PrimeTime® qPCR assays, in which the primers and probes are provided in a single-tube format.

Proximity Ligation

Life Technologies has created a new technology to systematically quantify proteins within a small sample by coupling antibody-mediated protein binding with qPCR quantification. According to Mark Shannon, Ph.D., senior staff scientist, “TaqMan® Protein Assays are an extension of proximity ligation assays (PLA™).

“In our system,” he went on to say, “the assay probes are target-specific antibodies that are conjugated to two different oligonucleotides through a biotin-streptavidin linkage. When the antibodies bind their target, the oligos come in proximity of each other. Addition of a connector oligonucleotide and DNA ligase creates a DNA amplicon, which is amplified in a qPCR reaction. The qPCR results correlate with the amount of protein in the sample.”

According to Dr. Shannon, this method for rapid quantification of proteins can be applied to formalin-fixed, paraffin-embedded (FFPE) and frozen samples. “The assays performed equivalently on both types of samples. This is important because there are vast archives of uncharacterized FFPE human tumor specimens.

“Often these are analyzed using immunohistochemistry, but that is much more labor intensive and much less quantitative. Thus, studies now can be conducted with greater ease and throughput with actual tumors. This will allow a better understanding of the protein profiles of cancers, and thus potentially identify new therapeutic biomarkers.”

Another benefit of the new technology is its ability to study interdependencies of proteins. “For example, we can use a model system to study the interplay of proteins in a disease pathway. As we knock down certain transcripts, we can monitor effects not only on the corresponding proteins but also on proteins in downstream pathways. Our technology enables a more systematic analysis of multiple proteins within a small sample that is highly sensitive and quantitative.”

The company initially provided six premade assay kits but recently began marketing an open-kit format to allow investigators to couple their own antibodies of interest.

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